Agonist and antagonist peptides of carcinoembryonic antigen (CEA)

ABSTRACT

A peptide comprising an agonist of a MHC Class I binding native sequence having amino acid substitution(s) and enhanced immunogenicity compared to the native sequence is described. Pharmaceutical compositions, peptide-immunoglobulin conjugates, kits and peptide-carrier molecules comprising such peptides also are described.

FIELD OF THE INVENTION

[0001] The present invention relates to the preparation and use ofpeptides that can act as agonists and antagonists of humancarcinoembryonic antigen (CEA). More specifically, the agonist peptideaccording to the present invention can be used as an immunogen, eitheralone, or in prime and boost protocols with other immunogens such asrV-CEA, for a variety of neoplastic conditions. These may includecolorectal cancer, lung cancer, pancreatic cancer, and breast cancer.Thus, the present invention also relates to the production and use ofvaccines against cancer. Peptide agonists according to the presentinvention can also be used to facilitate propagation of T cells, forexample, from vaccinated patients, for adoptive transfer studies.Peptide antagonists according to the present invention find utility insuppressing autoimmune responses, such as those involving T cells, whensuch responses occur in vaccinated patients. Thus, the present inventionalso relates to the production and use of vaccines against autoimmunediseases, especially those mediated by lymphocytes and other antigenpresenting cells.

BACKGROUND OF THE INVENTION

[0002] A major challenge of modern cancer immunotherapy is theidentification of cytotoxic T lymphocyte (CTL) epitopes from definedtumor-associated antigens (TAA) that promote lysis of tumor cells. Themajority of antigens on human cancers are not tumor specific and areoverexpressed in malignant cells as opposed to cells of normal tissues.Therefore, immunity to cancer in humans may rest mostly on thedevelopment of an effective immune response mainly directed toself-molecules qualitatively common to all cell types.

[0003] Human carcinoembryonic antigen (CEA) is a 180 kD glycoproteinexpressed on the majority of colon, rectal, stomach and pancreatictumors (1), some 50% of breast carcinomas (2) and 70% of lung carcinomas(3). CEA is also expressed in fetal gut tissue, and to a lesser extenton normal colon epithelium. The immunogenicity of CEA has beenambiguous, with several studies-reporting the presence of anti-CEAantibodies in patients (4-7) while other studies have not (8-10). CEAwas first described as a cancer specific fetal antigen in adenocarcinomaof the human digestive tract in 1965 (Gold, P. and Freeman, S. O. (1965)Exp. Med. 121:439-462). Since that time, CEA has been characterized as acell surface antigen produced in excess in nearly all solid tumors ofthe human gastrointestinal tract. The gene for the human CEA protein hasbeen cloned. (Oikawa et al (1987) Biochim. Biophys. Res. 142:511-518;European Application No. EP 0346710).

[0004] Recently, the first evidence was reported of a human CTL responseto CEA (11). This CAP1 peptide showed the highest level of T2 cellbinding among the various CEA peptides tested with stimulation of the Tcells resulting in the generation of cytotoxic T cell lines. We haveidentified a 9-mer peptide, designated CAP1 (with the sequenceYLSGANLNL) (SEQ. ID NO: 1), on the basis of binding to HLA-A2, and theability to generate specific CTL from peripheral blood mononuclear cells(PBMC) from carcinoma patients immunized with a recombinant vacciniavirus expressing CEA (rV-CEA). For example, peripheral blood lymphocytes(PBLs) from 5 patients showed signs of T cell response to CAP1 peptideafter immunization with rV-CEA. Two other laboratories have sincegenerated CAP1 specific CTL in vitro employing peptide pulsed dendriticcells as antigen presenting cells (APC) (12). It has also recently beenreported (13) that CAP1 specific CTL can be generated from PBMC fromcarcinoma patients immunized with the avipox recombinant ALVAC-CEA.Several groups have also reported the generation of anti-CEA antibodiesand CEA specific proliferative T cell responses following immunizationwith either an anti-Id to an anti-CEA monoclonal antibody (MAb) (14),recombinant CEA protein (15), or rV-CEA (16).

[0005] Several investigators have introduced CTL to tumor associated andviral antigens by in vitro stimulation of PBMC with an immunodominantpeptide. Recent work with the gp100 melanoma antigen (17-19), an HIVpolymerase peptide (20) and the papilloma virus tumor antigen E6 (21)demonstrated enhanced immunogenicity after modifications to the peptidesequences. In these studies, replacements were at anchor positions andwere intended to increase binding to murine or human MHC antigens. Thisapproach was based on a demonstrated correlation between immunogenicityand peptide binding affinity to class I MHC (major histocompatibilitycomplex) molecules for viral antigen epitopes (22).

[0006] Previous investigators have also worked with fragments of CEA.Thus, Shively (1989), in a European patent publication (EP No. 0343946A2) reports a number of CEA fragments that include a unique epitope (asdefined by its reactivity with an antibody). The latter CEA fragment is177 amino acid residues long and contains the 9-mer sequence of CAP1.However, no shorter CEA fragments that include the CAP1 sequence weredescribed.

[0007] In sum, the use of rV-CEA alone as an agent for boosting theCEA-specific immune response of rV-CEA suffers from the drawback ofstimulating an immune response to vaccinia virus. However, the novelcombination of rV-CEA and CAP1 suggested itself to us as a “secondgeneration protocol” for the treatment of cancer patients.

[0008] It is an accepted principle that when an immunogenic peptide ismodified in a conserved manner (e.g., a hydrophobic amino acid issubstituted with a hydrophobic amino acid) the modified peptide islikely to have similar immunogenic activity based upon the maintenanceof the molecule's shape, charge and hydrophobic character.

[0009] More specifically, a study by Madden (33) has identified specificamino acid preferences in peptides for MHC-complexing, a precursor stepto T cell recognition. Madden as well as other investigators (31)suggest that specific amino acid positions in peptides are available forT cell recognition.

[0010] Skipper et al. (40) describes the identification andcharacterization of a naturally-occurring peptide epitope of tyrosinase,wherein the peptide sequence differs from that which is predicted fromthe DNA. This modified peptide is recognized by tyrosinase-specifichuman cytotoxic T-lymphocytes (“CTL”) more effectively than the directtranslation product and is the only one of the two peptides to bepresented by HLA-A2.1 molecules on the cell surface. The modification isa substitution of an asparagine with an aspartic acid. The authorspropose that the asparagine is N-glycosylated in the endoplasmicreticulum during protein synthesis and is deamidatedpost-translationally.

[0011] In the case of CAP1, the primary and secondary anchors atpositions 2, 9, and 1 are already occupied by preferred amino acids andso a different approach was taken to improve peptide immunogenicity byattempting to enhance its ability to bind to the TCR. It appeared to usthat by altering amino acid residues expected to contact the TCR onecould generate an analog of CAP1 with substitutions at non-MHC anchorpositions. Such an analog might then represent a T cell enhancer agonistcapable of stimulating CTL more efficiently than the native peptide.Previous results supported the concept that some peptide analogs couldact as T cell antagonists by inhibiting responses to the antigenicpeptide (23-29). Such inhibition was shown to be TCR specific and couldnot be explained by competition for peptide binding to the MHC protein.Analogously, a peptide enhancer agonist would be an analog thatincreased the effector function without accompanying increases in MHCbinding. We therefore sought to increase CAP1 immunogenicity byanalyzing panels of analogs containing single amino acid substitutionsto residues we predicted would interact with the T cell receptor (TCR)of CAP1-specific CTL. The present invention relates to the constructionof a novel T cell enhancer agonist for the CAP1 peptide, the first suchexample for a human CTL epitope.

SUMMARY OF THE INVENTION

[0012] The present invention relates to the identification of peptideswhich are single or double amino acid changes from the CAP-1 peptidesequence. The CAP-1 peptide has been identified as a highly immunogenicepitope of the carcinoembryonic antigen (referred to herein as “CEA”),which is capable of stimulating CEA-specific cytolytic T-cell (“CTL”)responses. CEA is a cell surface antigen found in abundance on severaltypes of cancer cells. Thus, peptides of CEA capable of stimulating acytolytic CTL response, such as CAP-1 are potential immunogens for usein cancer immunotherapy.

[0013] Some of the peptides of the present invention are agonists ofCAP-1 and CEA; that is, they facilitate the interaction between theMHC-complex of the antigen-presenting cell and the T-cell receptor(“TCR”) complex of the T-cell. Thus, these peptides can.serve asimmunogens to treat and/or vaccinate patients with CEA-expressingcancers. Also, these peptides may be used to stimulate T-cells inculture for adoptive transfer of T-cells to cancer patients. Four suchpeptides have amino acid sequences:

[0014] (1) YLSGADLNL (Agonist CAP1-6D) (SEQ. ID NO: 2);

[0015] (2) YLSGADINL (Agonist CAP1-6D, 7I) (SEQ. ID NO: 3);

[0016] (3) YLSGANINL (Agonist CAP1-7I) (SEQ. ID NO: 4); and

[0017] (4) YLSGACLNL (agonist CAP1-6C) (SEQ. ID NO.: 5) .

[0018] The underlined amino acids identify the amino acids changes fromthe CAP-1 peptide sequence. Peptides CAP1-6D and CAP1-6D, 7I areespecially preferred peptides according to the present invention andhave enhanced activity as compared to CAP-1 activity. Peptides CAP1-7Iand CAP1-6C have activity similar to CAP-1.

[0019] Other peptides according to the present invention function asantagonists of CEA; that is, they reduce or eliminate CEA-specificT-cell activation and killing which occur through interactions of theMHC-peptide complex and TCR complex.

[0020] The present invention encompasses kits comprising an agonistpeptide and a vector comprising a gene encoding CEA or a recombinantlyproduced CEA protein. Moreover, the kit may include an immunostimulatorymolecule.

[0021] The present invention also encompasses kits comprising anantagonist peptide alone or in combination with an immunosuppressiveagent.

[0022] Another object of the present invention is a pharmaceuticalcomposition comprising one or more agonist peptides alone or incombination with an immunostimulatory molecule and a pharmaceuticallyacceptable carrier.

[0023] Another object of the present invention is a pharmaceuticalcomposition comprising one or more antagonist peptides alone or incombination with an immunosuppressing agent and a pharmaceuticallyacceptable carrier.

[0024] The present aspect of the present invention is a nucleic acidsequence encoding at least one agonist peptide or encoding at least oneantagonist peptide.

[0025] Another aspect of the invention is a vector comprising a nucleicacid sequence encoding at least one agonist peptide or a nucleic acidsequence encoding at least one antagonist peptide and host cellscomprising such vectors.

[0026] Another aspect of the present invention relates to the use ofthese peptides in cancer immunotherapy. The agonist peptides are usefulin stimulating a cytolytic immune response to CEA, resulting tumorreduction and/or prevention. Accordingly, the present invention alsorelates to a method of treating cancer patients with the peptides aswell as a cancer vaccine. The antagonist peptides are useful in methodsof controlling autoimmune response to CEA or CAP-1.

[0027] Yet another aspect of the present invention is an agonist-pulsedantigen presenting cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1A-1D: Effect of single amino acid substitutions in CEA CAP1peptide on lysis by CEA CTL T-Vac8 C1R-A2 cells were labeled with ¹¹¹Inand incubated for 1 hour in round bottom wells (2,000/well) with eachsubstituted peptide at 1 (solid), 0.1 (open) and 0.01 (hatched) μg/ml.T-Vac8 CTL were added at E:T=1.45:1 and isotope release was measuredafter 4 hours. Spontaneous release was determined for each peptide at 1μg/ml. All assays were performed in triplicate. FIGS. 1A-1D depictsubstitutions at positions p5 through p8, respectively. Amino acids aredesignated by the single letter code; the amino acid encoding the nativeCAP1 sequence is indicated in each figure and along the right-handmargin.

[0029] FIGS. 2A and 2B: CAP1 and analogs show different sensitivity toCEA CTL T-Vac8 cytotoxicity FIG. 2A T2 and FIG. 2B C1R-A2 target cellswere labeled with ⁵¹Cr and incubated in round-bottomed 96 well plates(10,000/well) with CAP1 (◯) or substituted peptides CAP1-6D □) orCAP1-7I (⋄) at the indicated concentrations. After 1 hour, T-Vac8 CTLwere added at E:T=2.5:1 and isotope release was determined after 4hours. All assays were done in triplicate. NCA571 (Δ) is a 9-mer peptideobtained after optimal alignment of CEA with the related gene NCA (11).

[0030]FIG. 3: Effect of single amino acid substitutions in CAP1 peptideon binding to and stability of HLA-A2 complexes T2 cells were collectedin serum free medium then incubated overnight (10⁶ well) with peptidesCAP1(◯), CAP1-6D (□), or CAP1-7I (⋄) at the indicated concentrations.Cells were collected and assayed for cell surface expression offunctional HLA-A2 molecules by staining with conformation sensitive MAbBB7.2, HLA specific antibody W6/32 (not shown) and isotype control AbMOPC-195 (not shown). Mean fluorescent intensity was determined on alive, gated cell population. Figure insert: Cells were incubated withpeptide at 100 μg/ml overnight, then washed free of unbound peptide andincubated at 37° C. At the indicated times, cells were stained for thepresence of cell surface peptide-HLA-A2 complexes. The error barsindicate SEM for two experiments.

[0031] FIGS. 4A and 4B: CTL generated from apparently healthyindividuals with CAP1-6D peptide recognize CAP1 and CAP1-6D CTL lines(designated T-N1 and T-N2) were generated with CAP1-6D and were assayedfor peptide specificity. T-N1 was assayed after 5 cycles of stimulationat an effector to target ratio of 20:1 (FIG. 4A). T-N2 was assayed after10 cycles at an effector to target ratio of 15:1 (FIG. 4B). ⁵¹Cr-labeledC1R-A2 targets (5,000/well) were incubated with the indicated amount ofCAP1 (∘) or CAP1-6D (□) peptide. After 4 hours the amount of isotoperelease was determined in a gamma counter. Values were determined fromtriplicate cultures.

[0032] FIGS. 5A and 5B: CAP1-6D. but not CAP1 generated T cell linesfrom apparently healthy donors recognize tumor cells expressingendogenous CEA CAP1-6D generated T-N2 CTL (FIG. 5A) and T cellsgenerated with native CAP1 (FIG. 5B), were assayed after 9 cycles of invitro stimulation against tumor targets SW480 and SW1463 (CEA⁺, HLA-A2⁺,◯ and

respectively), SKmel24 (CEA⁻, −A2⁺, □) and K562 (⋄). Tumor cells werecultured for 72 hours in the presence of γ-IFN to up regulate HLA. Cellswere trypsinized and labeled with ⁵¹Cr and incubated (5,000 cells/well)with T-N2 CTL at increasing effector to target ratios. Cultures wereincubated for 4 hours and the amount of isotope release determined in agamma counter. Values were determined from triplicate cultures.

[0033]FIG. 6: MHC-class 1 A2.1 restriction of CTL line (T-N2) derivedfrom CAP1-6D agonist CTL line T-N2 was used as an effector for the humancolon carcinoma SW837 target cell. SW837 is CEA positive and HLA-A2.1negative. SW837 were infected at an MOI of 10:1 with either arecombinant vaccinia containing the A2.1 transgene (□) or wild typevaccinia (Δ).

[0034] FIGS. 7A and 7B: CTL generated with CAP1-6D lyse CEA positive,HLA-A2 positive tumors: Effect of IFN upregulation The T-N1 CTLgenerated with CAP1-6D were assayed against various tumor cell lines:SW480 (CEA⁺ and HLA-A2^(+,) ◯), SW1116 (CEA⁺ but −A2⁻, □) and CaOV3(CEA⁻ but −A2⁺, ⋄). Tumor cells were cultured 72 hours in the absence(FIG. 7A) or presence (FIG. 7B) of γ-IFN, trypsinized and labeled with⁵¹Cr then incubated (5,000 cells/well) with T-N1 CTL at increasingeffector to target ratios. Cultures were incubated for 4 hours and theamount of isotope release determined in a gamma counter. Values weredetermined from triplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The invention is an peptide agonist of the native CEA epitope,CAP-1 (SEQ. ID NO: 1), as well as antagonists of SEQ. ID NO: 1. Theagonist is characterized by its ability to elicit antigen specificcytotoxic T lymphocytes which inhibit the growth or kill carcinoma cellsexpressing CEA or CEA epitopes. An antagonist of the present inventionserve to inhibit or prevent CEA specific immune responses. Such peptidesmay be used to shut off any unwanted immune responses to CAP-1 or CEA.One example for such use of an antagonist is to control any possibleautoimmune response that may occur during cancer immunotherapy, wherethe therapy has killed off tumor cells and begins to attack normal cellsexpressing CEA. In accordance with the present invention an antagonistwould advantageously prevent extensive damage to normal tissue.

[0036] The peptide agonists of the present invention comprise about 8-13amino acids, preferably 9-10 amino acids. In a preferred embodiment, theagonist peptide of the present invention comprises at least one aminoacid substitution at a non-anchor position. In one embodiment, theagonist comprises a sequence with a substitution at position 6 comparedto the native CAP-1 (SEQ. ID NO: 1). In another embodiment the agonistcomprises a sequence with an amino acid substitution at position 7compared to the native CAP-1 (SEQ. ID NO: 1). In yet another embodiment,the agonist comprises a sequence with an amino acid substitution atposition 6 and at position 7 compared to the native CAP-1. Thesubstituted amino acid serves to enhance the interaction of the TCRcomplex on the cytotoxic T lymphocytes with the peptide-MHC antigenligand complex. Such enhanced interaction results in greater effectorfunction by the cytotoxic T lymphocytes.

[0037] An example of a substitution includes Asp and Cys at position 6or an Ile at position 7.

[0038] In one embodiment, the peptide agonist comprises the followingamino acid sequence: Amino Acid Position 1 2 3 4 5 6 7 8 9 Native CAP-1Peptide Y L S G A N L N L (SEQ. ID NO: 1) Agonist Y L S G A D L N L(SEQ. ID NO: 2) Agonist Y L S G A D I N L (SEQ. ID NO: 3) Agonist Y L SG A N I N L (SEQ. ID NO: 4) Agonist Y L S G A C L N L (SEQ. ID NO: 5)

[0039] The agonist peptide of the present invention may be obtained byrecombinant DNA technology or by chemical peptide synthesis.

[0040] The agonist peptide may be formulated into a pharmaceuticalcomposition in combination with a pharmaceutically acceptable carrierfor use as an immunogen in a mammal, preferably a human. The compositionmay further comprise one or more other constituents to enhance theimmune response which include but are not limited to immunostimulatorymolecules such as interleukin 2, interleukin 6, interleukin 12,interferon gamma, tumor necrosis factor alpha, GM-CSF, B7.1, B7.2,ICAM-1, LFA-3, CD72, and cyclophosphamide.

[0041] The agonist peptide is administered to a mammal in an amounteffective in generating a CEA specific immune response, preferably acellular immune response. The efficacy of the mutant ras peptide as animmunogen may be determined by in vivo or in vitro parameters as areknown in the art. These parameters include but are not limited toantigen specific cytotoxicity assays, regression of tumors expressingCEA or CEA epitopes, inhibition of cancer cells expressing CEA or CEAepitopes, production of cytokines and the like.

[0042] At least one or more agonist peptides may be administered in adose of about 0.05 mg to about 10 mg per vaccination of the mammal,preferably about 0.1 mg to about 5 mg per vaccination. Several doses maybe provided over a period of weeks as indicated. In one embodiment adose is provided every month for 3 months. The agonist peptide may beadministered alone or in combination with adjuvants, incorporated intoliposomes (U.S. Pat. Nos. 5,643,599; 5,464,630; 5,059,421; 4,885,172),with cytokines, biological response modifiers, or other reagents in theart that are known to enhance immune response. Adjuvants include but arenot limited to RIBI Detox™, QS21, alum and incomplete Freund's adjuvant.In one embodiment, the mutant ras peptide is administered in combinationwith Detox™ (RIBI Immunochem Research, Hamilton, Mont.). RIBI Detox™contains as active ingredients the cell wall skeleton from Mycobacteriumphlei and monophosphoryl lipid A from Salmonella minnesota R595 preparedas an oil-in-water emulsion with squalene and tween 80.

[0043] The agonist peptides may also be conjugated to helper peptides orto large carrier molecules to enhance the immunogenicity of the peptide.These molecules include but are not limited to influenza peptide,tetanus toxoid, tetanus toxoid CD4 epitope, Pseudomonas exotoxin A,poly-L-lysine, a lipid tail, endoplasmic reticulum (ER) signal sequenceand the like.

[0044] The peptides of the present invention may also be conjugated toan immunoglobulin molecule using art accepted methods. Theimmunoglobulin molecule may be specific for a surface receptor presenton tumor cells but absent or in very low amounts on normal cells. Theimmunoglobulin may also be specific for a specific tissue. Such apeptide-immunoglobulin conjugate allows for targeting of the peptide toa specific tissue and/or cell.

[0045] Another effective form of the agonist peptide for generating anpeptide specific immune response in a mammal is an agonistpeptide-pulsed antigen presenting cell. The antigen presenting cellsinclude but is not limited to dendritic cells, B lymphocytes, monocytes,macrophages and the like. In a preferred embodiment, the agonistpeptide-pulsed antigen presenting cell is a dendritic cell.

[0046] The invention also provides a method of generating CEA andagonist peptide specific cytotoxic T lymphocytes in vivo or in vitro bystimulation of lymphocytes from a source with an effective amount of aagonist alone or in combination with a immunostimulatory molecule and/oradjuvant or in a liposome formulation. The sources of lymphocytesinclude but are not limited to peripheral blood, tumor tissues, lymphnodes and effusions such as pleural fluid or ascites fluid and the like.

[0047] The CEA and agonist peptide specific cytotoxic T lymphocytes ofthe present invention are immunoreactive with CEA agonist or peptide.The cytotoxic T lymphocytes inhibit the occurrence of tumor cells andcancer and inhibit the growth or kill expressing tumor cells expressingCEA or eptiopes thereof or agonist expressing tumor cells. The cytotoxicT lymphocytes, in addition to being antigen specific, are MHC class Irestricted. In one embodiment the cytotoxic T lymphocytes are MHC classI HLA-A2 restricted. The cytotoxic T lymphocytes have a CD8⁺ phenotype.

[0048] Selected patients bearing carcinoma cells expressing CEA or CEAepitopes are vaccinated subcutaneously up to three times at monthlyintervals with DETOX™ adjuvant admixed with the appropriate peptideagonist may also be vaccinated carcinoma patients with autologousperipheral blood mononuclear cells pre-pulsed ex vivo with a peptideagonist alone or in combination with a peptide agonist. Anti-CEA T cellresponses are evaluated as measured by proliferation assays.

[0049] Vaccination with CEA agonist peptides of the present inventioninduces highly specific and systemic anti-CEA cellular immune responses.Moreover, the development of such MHC class I-restricted agonistpeptides has important implications for both active (i.e., vaccination)and passive (i.e., ex vivo expansion for cellular adoptive transfer)immunotherapies, which may be used for the induction and propagation ofspecific CD8⁺ CTL responses in cancer patients.

[0050] Patients with solid tumors expressing CEA or epitopes thereof,including but not limited to colon cancer, lung cancer, pancreas cancer,endometrial cancer, breast cancer, thyroid cancer, melanoma, oralcancer, laryngeal cancer, seminoma, hepatocellular cancer, bile ductcancer, acute myeloblastic leukemia, basal cell carcinoma, squamous cellcarcinoma, prostate cancer and the like benefit from immunization withthe agonist peptides. Patients amenable to treatment using the agonistpeptides of the present invention are those patients having tumors withCEA or CEA epitopes.

[0051] Peptides may be chemically synthesized under GMP conditions andpurified by HPLC to >95% purity and lyophilized. Pharmaceuticalcompositions are formulated by reconstituting the peptide with apharmaceutically acceptable carrier such as sodium chloride. In oneexample, each milliliter of solution contains 1500 μg of a agonistpeptide plus 9.0 mg sodium chloride.

[0052] When the agonist peptide is administered with an adjuvant it isdesirable to mix the peptide with the adjuvant shortly beforeadministration to a patient.

[0053] The agonist peptide may be administered to a patient by variousroutes including but not limited to subcutaneous, intramuscular,intradermal, intraperitoneal, intravenous and the like. In oneembodiment the agonist peptide is administered subcutaneously. Thepeptide may be administered at one or more sites to a patient. In oneembodiment, the peptide, alone or in combination with an adjuvant, isadministered into three sites subcutaneously, over the deltoids, thethighs and the abdomen.

[0054] In another method of generating an immune response, agonistpeptide-pulsed antigen presenting cells are administered to the patientin an amount effective to generate an antigen specific immune response.The antigen presenting cells include but are not limited to dendriticcells, B lymphocytes, monocytes, macrophages and the like. In oneembodiment, dendritic cells are isolated from a patient by methodsdescribed in Romani, N. et al (1994). The isolated dendritic cells arecultured in vitro with an agonist peptide for a period of about 0.5 toabout 3 hours and washed to remove non-bound peptide. The agonistpeptide-pulsed dendritic cells are transferred back into the patient ata concentration of about 10⁶ to about 10⁹ dendritic cells. Such aconcentration is effective in generating an immune response in thepatient including the generation of agonist peptide specific cytotoxic Tlymphocytes which are able to inhibit the growth or kill tumor cells.

[0055] The criteria for determining an anti-tumor response in theimmunized patient is as follows:

[0056] 1. Complete Remission (CR): Complete disappearance of allevidence of tumor and return of abnormal tests to normal levels for aminimum of 4 weeks.

[0057] 2. Partial Response (PR): Decrease by at least 50% in the sum ofthe products of the perpendicular diameters of all measured lesions inthe absence of progression of any lesion nor the appearance of any newlesions for at least 4 weeks.

[0058] 3. Stable Disease (SD): Change in measurable disease too small tomeet the requirements for partial response or progression and theappearance of no new lesions for a period of at least 12 weeks. Theremay be no worsening of symptoms.

[0059] 4. Progressive Disease (PD) or Relapse: Any one of the criteriabelow must be met to be considered progressive disease:

[0060] Development of any new area of malignant disease (measurable orpalpable),

[0061] Increase (>25%) in any pretreatment area of measurable malignantdisease.

[0062] The immunological response to immunization with the agonistpeptides are assessed by in-vitro T cell proliferation assay and/or byin-vitro T cell cytotoxic assay before and after vaccination.

[0063] The present invention includes in vitro immunization for T cellproliferation and generation of cytotoxic T cell lines to the tumorspecific agonist peptide. In vitro cultivation of peptide specific Tcells from peripheral blood mononuclear cells (PBMC), lymph node tissue(LNT), or tumor infiltrating lymphocytes (TIL) with agonist peptide andIL-2 generates CEA and agonist peptide specific T cells. These T cellsare tested for cytotoxicity against agonist peptide primed APC(autologous EBV transformed B cells or autologous tumor cells) hasdescribed herein. Generated T cell clones are characterizedphenotypically by flow cytometry for express of CD3, CD4, and CD8.Agonist peptide specific cytotoxic lymphocytes may be adoptivelytransferred to a patient in order to inhibit or kill CEA or CEA epitopesexpressing tumor cells. Patients may then be reimmunized with agonistpeptide preferably in adjuvant.

[0064] Generally, between about 1×10⁵ and 2×10¹¹ cytotoxic T cells perinfusion are administered in, for example, one to three infusions ofabout 200 to about 250 ml each over a period of 30 to 60 minutes. Aftercompletion of the infusions, the patient may be treated with abiological response modifier such as interleukin 2 (IL-2). In the caseof IL-2, recombinant IL-2 is administered intravenously in a dose of720,000 IU per kilogram of body weight every eight hours. After adoptivetransfer of the antigen specific cytotoxic T cells into the patient, thepatient may be additionally treated with the agonist peptide used toprime the cytotoxic T cells, to further expand the T cell number invivo.

[0065] The invention encompasses a DNA sequence and variants thereofwhich encode an agonist peptide.

[0066] In one embodiment the DNA sequence encoding the agonist peptideis a variant of the DNA sequence comprising: (SEQ. ID No: 6) TAC CTT TCGGGA GCG AAC CTC AAC CTC (SEQ. ID No: 1) Tyr Leu Ser Gly Ala Asn Leu AsnLeu.

[0067] One variant of SEQ. ID No: 6 includes but is not limited to acodon ATC (Ile) in place of the codon, CTC (Leu at position 7). Anothervariant of SEQ. ID No: 6 includes but is not limited to a codon, TGT(Cys) in place of the codon, AAC (Asn at position 6).

[0068] In another embodiment, the DNA sequence encoding the agonistpeptide comprises: (SEQ. ID No: 7) TAC CTT TCG GGA GCG GAC CTC AAC CTC(SEQ. ID No: 2) Tyr Leu Ser Gly Ala Asp Leu Asn Leu

[0069] and variants thereof.

[0070] In yet another embodiment, the DNA sequence encoding the agonistpeptide comprises: (SEQ. ID No: 8) TAC CTT TCG GGA GCG GAC ATC AAC CTC(SEQ. ID No: 3) Tyr Leu Ser Gly Ala Asp Ile Asn Leu

[0071] or variants thereof.

[0072] Included in the ambit of the invention are conservativesubstitutions based on codon degeneracy provided the modificationresults in a functionally equivalent agonist peptide or a peptide withenhanced immunogenicity.

[0073] The invention further provides vectors and plasmids comprising aDNA sequence encoding an agonist peptide. The vectors include but arenot limited to E. coli plasmid, a Listeria vector and recombinant viralvector. Recombinant viral vectors including but not limited to orthopoxvirus, avipox virus, capripox virus, suipox virus, vaccinia,baculovirus, human adenovirus, SV40, bovine papilloma virus, and thelike comprising the DNA sequence encoding an agonist peptide.

[0074] Recombinant agonist peptide can be obtained using a baculovirusexpression system in accordance with the method of Bei et al J. Clin.Lab. Anal. 9:261-268 (1995). Recombinant viral vectors can beconstructed by methods known in the art such as U.S. Pat. No. 5,093,258;WO96/10419 Cepko et al Cell 37:1053-1062 (1984); Morin et al Proc. Natl.Acad. Sci USA 84:4626-4630 (1987); Lowe et al Proc. Natl. Acad. Sci USA84:3896-3900 (1987); Panicali & Paoletti, Proc. Natl. Acad. Sci USA79:4927-4931 (1982); Mackett et al, Proc. Natl. Acad. Sci USA79:7415-7419 (1982); WO 91/19803; Perkus et al Science 229:981-984(1985); Kaufman et al Int. J. Cancer 48:900-907 (1991); Moss Science252:1662 (1991); Smith and Moss BioTechniaues November/December, p.306-312 (1984); U.S. Pat. No. 4,738,846; Sutter and Moss Proc. Natl.Acad. Sci USA 89:10847-10851 (1992); Sutter et al Virology (1994); andBaxby and Paoletti Vaccine 10:8-9 (1992).

[0075] Host cells which may express the DNA encoding the agonist peptidecarried by vectors or plasmids are prokaryotic and eukoryotic host cellsand include but are not limited to E. coli, Listeria, Bacillus species,COS cells, Vero cells, chick embryo, fibroblasts, tumor cells, antigenpresenting cells and the like. When the host cell is an antigenpresenting cell, the host cell is an antigen presenting cell, the hostcell should additionally express an MHC class I molecule.

[0076] We recently reported (11) evidence of CTL responses to CEA inpatients immunized with rV-CEA. The 9-mer peptide CAP1 was employed toexpand CTL in vitro because of: (a) its strong binding to HLA-A2, and(b) its non-identity to other members of the CEA gene family expressedon normal tissues. CTLs were generated from post-immunization PBMC ofpatients while preimmunization blood of the same patients failed toproliferate. In addition, CAP1 pulsed dendritic cells stimulated invitro growth of −A2 restricted CTL from peripheral blood of unimmunizedcancer patients (12). Finally when CTL were generated in vitro bystimulation with dendritic cells encoding full-length CEA mRNA,cytotoxicity against CAP1 was higher than activity against six other −A2binding CEA peptides (S. Nair and E. Gilboa, personal communication orunpublished observation). Such results encourage the notion that CAP1 isan immunodominant epitope of the CEA molecule.

[0077] The present invention is intended to improve the immunogenicityof the CAP1 peptide by introducing amino acid substitutions atnon-anchor positions to form the agonist peptides of the presentinvention. When using T-Vac8 CTL as an effector, the analog CAP1-6Dsensitized target cells for lysis far better than CAP1 itself. Furtherstudies showed that cytolytic activity of a second −A2 restricted, CAP1specific CTL, T-Vac24, was as good or greater with CAP1-6D than withCAP1. These demonstrations of enhanced reactivity could not be explainedby improved presentation by class I MHC. Finally, CAP1-6D could be usedto stimulate CTL in vitro from PBMC of both carcinoma patients andnormal donors. Prior to the present invention, attempts to stimulateanti-CAP1 CTL from normal donors using this same methodology have beenunsuccessful. The present invention relates to stimulation of normaldonors with CAP1-6D as opposed to native CAP1 where stimulation with thenative sequence failed to produce specific cytotoxic activity. Incontrast, stimulation with CAP1-6D produced several CTL with specificanti-CAP1 peptide reactivity as well as anti-tumor reactivity. Thus, theanalog peptide CAP1-6D is capable of selecting a population of CAP1specific human CTL more efficiently than native CAP1. Such an agonistmight find applications in the design of T cell directed vaccinesagainst CEA-expressing carcinoma.

[0078] The present invention also relates to the more efficientgeneration and expansion of tumor specific T cells for adoptiveimmunotherapy. In recent years, much progress has been achieved incharacterizing the tumor associated antigen peptides that can bepresented to CTL by class I HLA antigens. In instances where mutationsgenerate neo-antigens such as point mutated ras (35, 36), p53 (37, 38)or β-catenin (39) vaccination strategies target the novel sequence underthe assumption that the immune system is not “tolerant” to an antigen ithas never seen. More recently it has been proposed that neo-antigens mayalso arise through post-translational deamidations (29, 40). However, inmany instances the intended targets of tumor therapy are not neoantigensbut rather normal oncofetal or differentiation antigens that areoverexpressed or ectopically expressed by malignant cells. Such is thecase for CEA (41). In such situations, models invoking “tolerance”predict that the immune system has encountered these antigens and isless able to respond to them. This classical picture has been challengedin recent years by numerous reports of immunity elicited tooverexpressed differentiation antigens, oncogenes, and tumor suppressorgenes (37, 38, 42-44). Nonetheless, it is often experimentally difficultto generate and expand T cells with desired anti-tumor activity and itis therefore desirable to devise new strategies for generating CTL.

[0079] Some class II binding-peptides have been described in whichsubstitutions enhance responses of murine and human Th clones withoutincreasing the binding to class II antigens (29, 45-47). Among humanclass I peptides, however, the only substitutions described for thegeneration of CTL were those that increase binding to HLA (17-20). Thesubstitutions in those studies were directed to residues at the primaryor secondary anchor positions that define the binding motifs to class IMHC antigens. Even substitutions directed to a non-anchor position (19)achieved their enhancing effect by increasing binding to HLA-A2. Theanalog CAP1-6D in the present report represents what appears to be adifferent class of substituted CTL peptides, agonists that enhancerecognition of the peptide-MHC ligand by the T cell receptor and producegreater effector function without increases in binding. To our knowledgethis is the first such enhancer agonist peptide described for a humanCTL.

[0080] The increased lytic susceptibility of targets in the presence ofCAP1-6D is unlikely to be due to better antigen presentation. Bindingexperiments show that HLA-A2 presents the native CAP1, and the analogsCAP1-6D and CAP1-71 approximately equally. Another possibility is thatCAP1-6D shows increased activity because it is presented by more thanone allele and T-Vac8 is promiscuous towards peptide-MHC complexes.However, T-Vac8, T-Vac24, and CTL derived from nonimmunized patientsshowed better lysis with CAP1-6D. Since HLA-A2 is the only class I MHCon the targets employed, the improved lysis cannot be accounted for byrecruitment of another class I MHC.

[0081] Since anti-CAP1 CTL from multiple donors demonstrate agonistcross reactivity it is possible that CAP1-6D could be used to stimulategrowth of CTL from numerous −A2 individuals. We are encouraged by thequite distinct differences between T-Vac8 and T-Vac24 in magnitude ofresponse to the agonist; this implies that each effector utilizesdifferent TCR gene segments and that nonetheless they can recognize boththe native sequence and the CAP1-6D substitution. The ability of CAP1-6Dto act as an agonist with T cells expressing different T cell receptorsclearly magnifies its therapeutic potential. Thus, the present inventionalso relates to stimulation with the agonist and subsequent generationof T cells that recognize the normal sequence in non-immunizedindividuals. Such individuals have presumably never encountered themodified sequence and since the agonist is more efficient at triggeringa T cell response, such agonists might be capable of selecting CTL morereadily than immunogens based on the native sequence.

[0082] For peptide-derived CTL to be useful therapeutic reagents it isessential to demonstrate that they can lyse tumor cells that expressendogenous antigen (48, 49). Previously (11), we had shown that tumorcells process CEA and present antigens recognized by CTL generated bystimulation with CAP1. In accordance with the present invention, CTLgrown from the normal donors by stimulation with CAP1-6D are alsocapable of recognizing allogeneic CEA-positive, HLA-A2 positive tumorcells. These T cells fail to recognize −A2 negative tumor cells or −A2positive cells that lack CEA expression.

[0083] We have also shown that CTL selected with the CAP1-6D agonist canbe maintained subsequently by stimulation with the native CAP1 sequence.This is an important finding since CTL in patients, whether establishedin vivo through active immunization, or transferred adoptively after exvivo expansion, will likely only encounter the native sequence. Thisallows the CTLs to be maintained over an extended duration in vivo.

[0084] One of the original reasons for selecting and testing CAP1 wasits non-identity with other reported sequences in the human genome. Itwas therefore predicted that any immune responses attained would beunlikely to damage normal tissues bearing other antigens. For thisreason a similar search of protein databases was undertaken for thepeptides CAP1-6D and CAP1-7I and revealed that they are not reported ashuman sequences elsewhere in the Genebank (Genetics Computer Group,Madison, Wis.). However, two similar sequences, YLNVQDLNL (SEQ. ID No:9) and YLHDPEFNL (SEQ. ID No: 10), are reported for antigens fromAfrican swine fever virus and measles virus, respectively. Thesesequences fit the consensus motif for HLA-A2 and therefore allowinfected individuals to express cross-reacting antigens to CAP1. Oneinteresting possibility is that the presence of anti-CAP1 CTL in somepatients represents an example of epitope mimicry (50).

[0085] Two recent reports suggest that modified asparagine residuesmight enhance the immunogenicity of class I MHC peptides. Skipper et al.(40) used CTL generated in mixed lymphocyte tumor cell cultures toidentify antigens in extracts of melanoma cells. One antigenic peptidewas identical at 8 of 9 positions to a sequence from tyrosinase, with anasparagine to aspartic acid replacement at position 3. When tested usingsynthetic peptides, the CTL were more active against the aspartic acidpeptide than against the peptide containing the genetically predictedasparagine. These authors speculate that post-translational deamidationscan generate antigenic peptides from normal differentiation antigens.Recently, Chen et al. (51) reported generating murine CTL to astabilized succinimide derivative of an asparagine-containing antigenicpeptide. Although these CTL could kill targets pulsed with the naturalasparagine peptide, they did so with less sensitivity. They raise thepossibility that deamidation of proteins in vivo and in vitro canproduce transient succinimide intermediates that represent alteredself-ligands capable of eliciting an immune response. At the otherextreme, Kersh and Allen (52) replaced a TCR contact asparagine withaspartic acid in a hemoglobin peptide and abolished responsiveness to amurine Th clone. Presently we cannot exclude the possibility that theenhanced reactivity of CAP1-6D is due to deamidation of the nativesequence which in turn primes the response that we detect with CAP1.However, our repeated inability to raise anti-CAP1 CTL frompre-immunized PBMC of the same patients from whom we generatedpost-immunization CTL, argues against this. Also, putative deamidationscould not account for the recognition of other analogs such as CAP1-6Cor CAP1-7I by T-Vac8 CTL. Instead it seems more reasonable that T cellreceptors from both T-Vac8 and T-Vac24, as well as the new linesdescribed here, can recognize some deviation from the native CAP1sequence.

[0086] In summary, synthesis of analogs of an immunodominant CEA peptidewith amino acid substitutions at positions predicted to potentiallyinteract with the T cell receptor allowed us to identify an enhanceragonist. This agonist was recognized by two different CEA CTL andincreases the activity of one of them by 2-3 orders of magnitude. Theagonist was also able to stimulate growth of CTL from peripheral bloodof non-immunized normal donors with far greater facility than the nativepeptide sequence. Most important, the CTL generated using the enhanceragonist was able to recognize and lyse targets presenting the nativesequence, including tumor cell lines expressing endogenous CEA. Inaccordance with the present invention, characterization of this enhanceragonist peptide facilitates more aggressive anti-tumor immunotherapieswhen employed as an immunogen in vivo, or for the ex vivo expansion ofautologous anti-tumor CTL. The synthetic approach employed according tothe present invention is also useful in improving immunogenicity ofother peptide CTL epitopes.

MATERIALS AND METHODS

[0087] Peptides

[0088] A panel of single amino acid substitutions to positions p5through p8 of the CEA peptide CAP1 were made by f-moc chemistry usingpin technology (Chiron Mimotopes, Victoria, Australia). CAP1 (YLSGANLNL)and CAP1˜6D (YLSGADLNL), greater than 96% pure, were also made byMultiple Peptide Systems (San Diego, Calif.). Additional peptidesCAP1-7I and NCA571 were synthesized on an Applied Biosystems 432Asynthesizer and were greater than 90% pure. by C18 reverse-phase HPLC.

[0089] Cell Lines

[0090] T-Vac8 (53) and T-Vac24 (11) are human CTL specific for the CEApeptide CAP1. These cell lines were generated by in vitro stimulation ofPBMC using CAP1 and IL-2, according to previously published methods(11). Briefly, post-immunization PBMC were from HLA-A2+ individuals withadvanced carcinoma that had been administered rV-CEA in a Phase I trial.PBMC were isolated on gradients of lymphocyte separation medium (OrganonTeknika, Durham, N.C.) and 2×10⁵ cells were placed in wells of sterile96 well culture plates (Coming Costar, Cambridge, Mass.) along with 50μg/ml peptide. After 5 days incubation at 37° C. in a humidifiedatmosphere containing 5% CO₂, supernatants were removed and replacedwith medium containing 10 U/ml human IL-2 (a gift of the Surgery Branch,NCI). Cultures were fed with IL-2 every 3 days for 11 days and thenrestimulated with irradiated (4000 rad) autologous PBMC (5×10⁵) andpeptide. Fresh IL-2 was provided every third day and subsequentrestimulations were done every 2 weeks. CTL are maintained in completeRPMI (GIBCO/BRL, Grand Island, N.Y.) medium with glutamine (GIBCO/BRL),penicillin, streptomycin and 10% pooled human AB serum (GeminiBioproducts, Inc., Calabasas, Calif.).

[0091] Cell line C1R-A2 (provided by Dr. W. Biddison, National Instituteof Neurological Disorders and Stroke, National Institutes of Health,Bethesda, Md.) is maintained in complete RPMI with 10% fetal bovineserum (FBS, Biofluids Inc., Rockville, Md.), glutamine, non essentialamino acids and pyruvate (Biofluids) and 1 mg/ml G418. Cell line174.CEM-T2 (provided by Dr. P. Creswell, Yale University School ofMedicine, New Haven, Conn.) is defective in endogenous peptideprocessing and is maintained in Iscove's-(GIBCO/BRL) with 10% FBS. BothC1R-A2 and T2 lines present exogenous peptides with HLA-A2.

[0092] CEA positive tumor cell lines SW480, SW1463, SW1116 and SW 837were obtained from the American Type Culture Collection (ATCC,Rockville, Md.) and passaged weekly in respective culture mediumdescribed in the ATCC catalog. The CEA negative melanoma line SKmel24(provided by Dr. S. Rosenberg, National Cancer Institute, NationalInstitutes of Health, Bethesda, Md.) was passaged weekly in RPMI 1640,10% FBS and 10 μg/ml gentamicin (Life Technologies). The CEA negativeovarian tumor CaOV3 was provided by Dr. R. Freedman (MD Anderson CancerCenter, Houston Tex.) and was cultured in RPMI with 15% FBS, glutamine,12 μg/ml insulin (Sigma, St. Louis, Mo.), 10 μg/ml hydrocortisone(Biofluids) and 10 μg/ml gentamicin. All tumor lines were trypsinizedwith Trypsin/versene (Biofluids) for 5-10 minutes prior to labeling withisotope for CTL assays. The highly sensitive natural killer (NK) targetK562 was obtained from ATCC and passaged weekly with RPMI 1640, 10% FBS.

[0093] Generation of CTL

[0094] T cell lines T-N1 and T-N2 were generated from PBMC of two normalHLA-A2 positive donors by in vitro stimulation with peptide as follows.For the first stimulation cycle, T cells were positively selected bypanning on CD3+ MicroCellector flasks (Applied Immune Sciences, SantaClara, Calif.). CD3+ cells (3×10⁶) were cultured with 10⁶ 174.CEM-T2cells that were previously infected with vaccinia virus expressing humanB7 at a multiplicity of infection of 10, pulsed with 50 μg/ml CAP1 orCAP1-6D peptide and 2 μg/ml human β2 microglobulin (Intergen, Purchase,N.Y.), and irradiated (10,000 rad). Cultures were incubated at 37° C. ina humidified atmosphere containing 5% Co₂, in T25 flasks in RPMI with10% human serum, 2 mM glutamine, and 10 μg/ml gentamicin in a totalvolume of 10 ml with 2×10⁷ irradiated (2500 rads) autologous PBMC asfeeder cells. After 24 hours in culture 10 U/ml hulL-2 and 0.1 ng/mlrIL-12 (R & D Systems, Minneapolis, Minn.) were added. After 9 days inculture, cells were restimulated using irradiated (10,000 rads)autologous EBV-B cells preincubated with 25 μg/ml peptide at a ratio of2.5:1 stimulator cells to T cells, and IL-2 and IL-12 were again added24 hours later. Peptide concentration was halved with each subsequentstimulation cycle until a final concentration of 3.12 μg/ml wasachieved.

[0095] In addition, CTL were generated from post-immunization PBMC ofcancer patient Vac8 by stimulation with CAP1-6D according to alreadypublished procedures (11).

[0096] CTL Assay

[0097] Target cells were labeled with ⁵¹Cr or ¹¹¹In, then incubated at2,000-10,000 per well with or without peptides in round bottommicrotiter plates (Corning Costar). One hour later, T cells were added.Supernatants were harvested (Skatron, Inc., Sterling Va.) after 4 hourand isotope release was measured. All assays were performed intriplicate and percent specific release was calculated according to:$\frac{\text{(observed release-spontaneous release)}}{\text{(maxium release-spontaneous release)}} \times 100$

[0098] where spontaneous release is obtained by omitting the T cells,and maximum release is obtained by adding 1% Triton ×100.

[0099] Binding Assay

[0100] Binding of peptides to HLA-A2 was evaluated by incubation withprocessing defective 174.CEM-T2 cells and measuring the stability ofcell surface peptide-A2 complexes (30). Briefly, cells were harvestedand washed with serum-free RPMI then incubated overnight at 1-2×10⁶cells/well with various concentrations of peptides. The next day, cellswere collected, washed in PBS with Ca²⁺, Mg²⁺ and 5% FBS, then dividedinto aliquots for single color flow cytometric analysis. Cells wereincubated 1 hour on ice without antibody, with anti-A2 antibody A2,69(One Lambda, Inc., Canoga Park, Calif.) or with isotype-matched controlantibody UPC-10 (Organon Teknika) then washed and stained 1 hour withfluorescein-isothiocyanate (FITC) goat anti-mouse Ig (SouthernBiotechnology Associates, Birmingham, Ala.). Cell surface staining wasmeasured in a Becton Dickinson flow cytometer (Mountain View, Calif.)and the mean fluorescence intensity (MFI) for 10,000 live cells wasplotted against peptide concentration.

[0101] TCR Chain Usage

[0102] T-N1 CTL were cultured as described for 5 cycles of antigenicstimulation using the CAP1-6D analog. The line was then split andduplicate cultures were maintained either with CAP1 or CAP1-6D for 5additional stimulation cycles. Ficoll-purified T cells (5×10⁵) werestained with a panel of 19 anti-Vβ and 2 anti-Vα murine monoclonalantibodies to human αβ T cell receptor variable regions. Cells wereincubated with 10 μg/ml of purified antibodies for 30 minutes at 4° C.The unlabeled monoclonals used were: Vβ3.1 clone 8F10, Vβ5(a) clone 1C1,Vβ5(b) clone W112, Vβ5(c) clone LC4, Vβ6.7 clone OT145, Vβ8(a) clone16G8, Vβ12 clone S511, Vβ13 clone BAM13, Vα2 clone F1 and Vα12.1 clone6D6 (T Cell Diagnostics, Woburn, Mass.) and Vβ18 (Immunotech, Westbrook,Me.). Cells were stained with 10 μg/ml of FITC-labeled goat anti-mouseIgG antibody (Southern Biotechnology Associates) for 30 minutes in thedark. Directly labeled monoclonals were: FITC-labeled Vβ11, Vβ21.3,Vβ13.6, Vβ14, Vβ16, Vβ17, Vβ20 and Vβ22 and PE-labeled Vβ9 and Vβ23(Immunotech). Cells were fixed with 1% paraformaldehyde, washed withFACSFlow buffer (Becton Dickinson) and analyzed using a Becton Dickinsonflow cytometer.

EXAMPLES CAP1 Substituted Peptides

[0103] Several factors were considered in deciding which positions toexamine for effects on T cell activity. Sequencing and mappingexperiments have defined a binding motif in which position 2 and theC-terminal (position 9 or 10) are critical for peptide presentation byHLA-A2 (for review, see 31). In addition, Tyr at position 1 has beenidentified as an effective secondary anchor (20, 32). Since the CEApeptide CAP1 already has the preferred amino acids at these threepositions these residues were not altered. Instead, we focused attentionon residues predicted to interact with the TCR in the hope of findinganalogs that would stimulate human CAP1-specific cytotoxic T cells.X-ray crystallographic studies of several peptides bound to solubleHLA-A2 suggest that all binding peptides assume a common conformation inthe peptide binding groove (33). When five model peptides were examined,residues 5 through 8 protrude away from the binding groove and arepotentially available for binding to a TCR. Therefore a panel of 80 CAP1analog peptides was produced in which the residues at positions 5through 8 (p5-p8) were synthesized with each of the 20 natural aminoacids. The peptides are designated CAP1-pAA, where p refers to theposition in the peptide and AA refers to the replacement amino acid,using the single letter amino acid code; i.e., CAP1-6D in which position6 is occupied by aspartic acid.

Enhanced CTL Sensitivity of Targets to CAP1-6D Analog

[0104] The effects of these amino acid substitutions on potential TCRrecognition was studied using a CAP1 specific, HLA-A2 restricted humanCTL line designated T-Vac8. Briefly, T-Vac8 was generated as describedin Materials and Methods by in vitro peptide stimulation of PBMC from apatient that had been administered rV-CEA. For initial screening, T-Vac8was used in a cytotoxicity assay to measure ¹¹¹In release from labeledC1R-A2 cells incubated with each member of the peptide panel (at threepeptide concentrations). Spontaneous release from the targets (in theabsence of T-Vac8) was determined for each individual peptide.

[0105] The results are presented in FIGS. 1A through 1D. Of the 80single amino acid substitutions, most failed to activate cytotoxicity ofT-Vac8. However, six independent substitutions preserved reactivity. Atposition 5, three analogs CAP1-5F, CAP1-5I and CAP1-5S providedstimulation, albeit at reduced levels compared to CAP1 itself. Atposition 6 the substitutions CAP1-6C and CAP1-6D activated T-Vac8cytotoxicity and seemed to be equal to or better than CAP1 since theywere more active at the intermediate (0.1 μg/ml) peptide concentration.At position 7 analog CAP1-7I also appeared to be active. Finally, atposition 8, no analogs were able to sensitize targets to lysis byT-Vac8. The two most active analogs (CAP1-6D and CAP1-7I) were thenanalyzed in detail, omitting CAP1-6C due to concern for disulfideformation under oxidizing conditions.

[0106] Purer preparations (90-96% pure) of native CAP1 and the analogsCAP1-6D and CAP1-71 were synthesized and compared in a CTL assay over awider range of peptide concentrations, using two different cell lines astargets (FIGS. 2A and 2B). Employing T2 cells analog CAP1-6D was atleast 10² times more effective than native CAP1. CAP1-6D lytic activitywas at ½ maximum at 10⁻⁴ μg/ml (FIG. 2A). In contrast, the CAP1-71analog and the native CAP1 sequence were comparable with each other overthe entire range of peptide titration and showed half maximal lysis at10⁻² μg/ml. Employing the C1R-A2 cells as targets, CAP1-6D was similarlybetween 10² and 10³ more effective in mediating lysis than CAP1 (FIG.2B).

[0107] The CAP1-6D peptide was also tested using a second CEA-specIfic Tcell line, T-Vac24 (11). This line was generated from rV-CEA postvaccination PBMC of a different carcinoma patient by in vitrostimulation with the native CAP1 peptide; in contrast to predominantlyCD8+ T-Vac8, T-Vac24 has a high percentage of CD4+CD8+ double positivecells (11). In a 4 hr ¹¹¹In release assay employing T-Vac24, CAP1-6D wasslightly more effective (30% lysis) than the native CAP1 sequence (20%lysis); although the differences were not as pronounced as with T-Vac8,the increased sensitivity to the analog was seen in three separateexperiments. The analog peptide clearly engaged the lytic apparatus of asecond CAP1 specific CTL.

Analogs and Native Peptide Show Identical Presentation by HLA-A2

[0108] The increased effectiveness of CAP1-6D in CTL assays could be dueto better presentation by the target. The most active CAP1 analogs weretested for binding to HLA-A2 by measuring cell surface HLA-A2 in. thetransport-defective human cell line T2. When compared over a 4-log rangeof concentrations, native CAP1 and the two analogs CAP1-6D and CAP1-7Iall presented equally on T2 cells (FIG. 3). In addition, dissociationexperiments indicate that the HLA-A2 complexes that form with the 3peptides show no appreciable differences in stability (FIG. 3—insert).When peptide-pulsed T2 cells were washed free of unbound peptide, thehalf lives of cell surface peptide-A2 complexes were 12.5 hrs (CAP1),9.7 hrs (CAP1-6D), and 10.8 hrs (CAP1-71). If anything, the complexformed with the agonist peptide seems slightly less stable. Since thereare no differences in binding to HLA-A2, the improved. effectiveness ofCAP1-6D in the CTL assays appears to be due to better engagement by theT cell receptor, a behavior characteristic of an enhancer agonistpeptide.

Human CTL Generated With CAP1-6D also Recognize Native CAP1

[0109] The CAP1-6D agonist might be useful in both experimental andclinical applications if it can stimulate growth of CEA-specific CTLfrom patients with established carcinomas. In one experiment, postrV-CEA immunization PBMC from cancer patient Vac8 (the same rV-CEApatient from whom T-Vac8 CTL were established) were stimulated in vitrowith CAP1-6D and after 5 rounds of stimulation were assayed for CTLactivity against targets coated with CAP1 or CAP1-6D. This new linedemonstrated peptide-dependent cytotoxic activity against target cellscoated with either CAP1-6D or native CAP1 (Table 1).

[0110] Post immunization PBMC from patients Vac8 and Vac24 were alreadyshown to produce CTL activity when stimulated with CAP1 whilepreimmunization PBMC were negative (11, 34). Moreover, previous attemptsto stimulate CTL activity from healthy, non-immunized donors with theCAP1 peptide were unsuccessful. To test if the agonist peptide is indeedmore immunogenic than native CAP1 we attempted to generate CTL fromhealthy, non-immunized donors using CAP1-6D. HLA-A2+ PBMC fromapparently healthy individuals were stimulated in vitro either with CAP1or the CAP1-6D agonist. After 4 cycles of in vitro stimulation, celllines were assayed for specificity against C1R-A2 cells pulsed witheither CAP1 or CAP1-6D.

[0111] While stimulations with CAP1 or the CAP1-6D peptide produced Tcell lines, peptide specific lysis was only obtained in the linesgenerated with CAP1-6D. Two independent T cell lines from differentdonors were derived using CAP1-6D and were designated T-N1 and T-N2(FIG. 4A and FIG. 4B respectively). Both CTL lines lyse C1R-A2 targetspulsed with native CAP1 peptide. However, more efficient lysis isobtained using the CAP1-6D agonist. T-N1 CTL recognizes CAP1-6D at a3-10 fold lower peptide concentration than CAP1 and T-N2 recognizes theagonist 100 fold better than CAP1. In contrast, attempts to generate aCTL cell line from normal donors by stimulation with CAP1 resulted inlines with no peptide-dependent lysis and loss of the lines in earlystimulation cycles. Thus the attempts to generate T cell lines using thetwo peptides demonstrated the ability of CAP1-6D to act as an agonistnot only at the effector stage, in the lysis of targets, but also inselecting T cells that are presumably in low precursor frequencies.

[0112] To determine whether CTL established with the agonist could bemaintained on the native CAP1 sequence, T-N1 was cultured for 5 cyclesas described using CAP1-6D, then divided into duplicate culturesmaintained on the agonist or on CAP1. T-N1 continued to grow whenstimulated with either peptide and responded to both peptides in CTLassays. Phenotypic analysis of the TCR usage in T-N1 indicates that themajority of cells (71%) utilize Vβ12, with a minor population thatutilize Vβ5.3 (Table 2). The same pattern of TCR Vβ usage was observedafter switching the cells to CAP1 for 5 more stimulation cycles. This Vβusage pattern was distinct from that of T-Vac8. These data indicate thatthe agonist can select T cells that are probably in low precursorfrequency but that once selected, such CTL could be maintained with thenative CAP1.

CTL Generated with CAP1-6D Specifically Lysed CEA⁺, HLA-A2⁺ Tumor Cells

[0113] Studies were conducted to determine the ability of CTL generatedwith the enhancer agonist to lyse human tumor cells endogenouslyexpressing CEA. T-N1 and T-N2 were tested against a panel of tumor cellsthat are CEA⁺/A2⁺(SW480 and SW1463), CEA+/A2⁻ (SW1116) or CEA⁻/A2⁺(CaOV3 and SKmel24). A T cell line (T-N2) from the normal donor wastested for the ability to lyse tumor targets endogenously expressingCEA. T-N2 CTL generated with the agonist lysed tumor cells expressingboth CEA and HLA-A2 while exhibiting no titratable lysis of CEA⁻/A2⁺SKmel24 melanoma cells (FIG. 5A). No significant lysis of K562 wasobserved. In contrast, cell lines generated by stimulation with nativeCAP1 showed no detectable antitumor activity (FIG. 5B). The HLA-A2.1restriction of the T-N2 response to CEA positive tumor targets wasfurther demonstrated by the specific lysis of a CEA positive HLA-A2.1negative tumor cell, SW837 after infection with a vaccinia-A2.1construct (rV-A2.1). No lysis was observed when SW837 targets wereinfected with the control wild type vaccinia without the A2.1 transgene(FIG. 6)

[0114] The ability of a CTL line (T-N1) derived from a second donor tokill carcinoma targets expressing endogenous CEA is shown in FIGS. 7Aand 7B. T-N1 specifically lysed SW480 tumor cells. This is dramaticallyenhanced to 79% lysis by pretreatment of the tumor cells with IFN-γ, atreatment that increases the cell surface density of both HLA-A2 andCEA. The specificity of T-N1 killing is demonstrated by its inability tolyse CEA⁻/A2⁺ tumors such as the ovarian derived tumor CaOV3, themelanoma tumor SKmel24, or the NK target K562. Finally, restriction byHLA-A2 is demonstrated by the failure of T-N1 to lyse CEA⁺/A2⁻ SW1116tumor cells (FIG. 7A), even after IFN-γ treatment (FIG. 7B) TABLE 1 CTLgenerated by stimulation with the CAP1-6D analog from PBMC of an HLA-A2patient immunized with rVCEA % Lysis Effector/target ratio no peptideCAP1 CAP1-6D   25:1  10% 41% 40% 6.25:1 0.5% 38% 46%

[0115] T cells were assayed after 5 in vitro stimulations. Cytotoxicactivity was determined in 4 hour release assay with peptide at 25μg/ml. TABLE 2 TCR usage of CTL line established on CAP1-6D agonistT-N1^(b) T-N1^(c) TCR usage^(a) % positive MFI % positive MFI Vβ12 71 8370 83 Vβ5.3 18 47 20 57 Vβ3.1 6 48 8 46 Vβ8 3 30 6 26 Vβ13.6 2 19 3 39Vβ12.1 3 43 3 40

[0116] This invention has been described in detail including preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements thereon without departing from the spiritand scope of the invention.

[0117] References referred to are incorporated herein by reference.

References

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1 12 1 9 PRT Homo sapiens 1 Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5 2 9PRT Homo sapiens 2 Tyr Leu Ser Gly Ala Asp Leu Asn Leu 1 5 3 9 PRT Homosapiens 3 Tyr Leu Ser Gly Ala Asp Ile Asn Leu 1 5 4 9 PRT Homo sapiens 4Tyr Leu Ser Gly Ala Asn Ile Asn Leu 1 5 5 9 PRT Homo sapiens 5 Tyr LeuSer Gly Ala Cys Leu Asn Leu 1 5 6 27 DNA Homo sapiens 6 tacctttcgggagcgaacct caacctc 27 7 27 DNA Homo sapiens 7 tacctttcgg gagcggacctcaacctc 27 8 27 DNA Homo sapiens 8 tacctttcgg gagcggacat caacctc 27 9 9PRT Homo sapiens 9 Tyr Leu Asn Val Gln Asp Leu Asn Leu 1 5 10 9 PRT Homosapiens 10 Tyr Leu His Asp Pro Glu Phe Asn Leu 1 5 11 27 DNA Homosapiens 11 tacctttcgg gagcgaacat caacctc 27 12 27 DNA Homo sapiens 12tacctttcgg gagcgtgtct caacctc 27

We claim:
 1. A peptide comprising an agonist of a native sequence:YLSGANLNL (Seq. ID No: 1) |   | 123456789


2. The peptide according to claim 1 wherein the agonist varies in anamino acid substitution at position 6 from Seq. ID No:
 1. 3. The peptideaccording to claim 1 wherein the agonist varies in an amino acidsubstitution at position 7 from Seq. ID No:
 1. 4. The peptide accordingto claim 1 wherein the agonist varies in an amino acid substitution atposition 6 and position 7 from Seq. ID No:
 1. 5. The peptide accordingto claim 1 containing YLSGADLNL (Seq. ID No: 2), YLSGADINL (Seq. ID No:3), YLSGANINL (Seq. ID No: 4), YLSGACLNL (Seq. ID No: 5), or combinationthereof.
 6. A peptide consisting of the amino acid sequence YLSGADLNL(Seq. ID No: 2), YLSGADINL (Seq. ID No: 3), or YLSGANINL (Seq. ID No:4), YLSGACLNL (Seq. ID No: 5).
 7. A pharmaceutical compositioncomprising the peptide according to claim 1 and a pharmaceuticallyacceptable carrier.
 8. The pharmaceutical.composition according to claim7 further comprising an immunostimulatory molecule.
 9. Thepharmaceutical composition according to claim 8 wherein theimmunostimulatory molecule is selected from the gproup consisting ofIL-2, B7.1, B7.2, ICAM-1, LFA-3, CD72, GM-CSF, TNFα, INFγ, IL-12, IL-6and combinations thereof.
 10. The pharmaceutical composition accordingto claim 7 further comprising an HLA class I molecule or a cellexpressing an HLA class I molecule.
 11. The pharmaceutical compositionaccording to claim 7 further comprising a chemotherapeutic drug,antibiotic, antiviral drug, antifungal drug, or cyclophosphamide. 12.The pharmaceutical composition according to claim 7 further comprisingan adjuvant.
 13. The pharmaceutical composition according to claim 12wherein the adjuvant is selected from the group consisting of alum,incomplete Freund's adjuvant, QS21, and Ribi Detox™.
 14. Apeptide-immunoglobulin conjugate comprising the peptide according toclaim 1 and an immunoglobulin molecule.
 15. The pharmaceuticalcomposition according to claim 7 wherein the peptide is incorporatedinto a liposome.
 16. A peptide-carrier molecule conjugate comprising thepeptide according to claim 1 conjugated to a carrier molecule.
 17. Thepeptide-carrier molecule conjugate according to claim 15 wherein thecarrier molecule is selected from the group consisting of influenzapeptide, tetanus toxoid, tetanus toxoid-CD4 epitope, Pseudomonasexotoxin A, poly-L-lysine, a lipid tail and an endoplasmic reticulumsignal sequence.
 18. A kit comprising the agonist peptide according toclaim 1 and a vector comprising a nucleic acid sequence encoding CEA.19. The kit according to claim 18 further comprising animmunostimulatory molecule.
 20. An isolated DNA comprising a nucleotidesequence encoding the peptide according to claim 1 or variants thereof.21. An isolated DNA encoding a peptide containing Seq. ID No: 2, Seq. IDNo: 3, Seq. ID No: 4, Seq. ID No: 5, or combinations thereof.
 22. Anisolated DNA comprising a nucleotide sequence of SEQ. ID No: 7 or
 8. 23.A vector comprising the DNA of claims 20, 21 or
 22. 24. The vectoraccording to claim 23 wherein the vector is an E. coli plasmid, aListeria vector, an orthopox virus, avipox virus, capripox virus, suipoxvirus, vaccinia virus, baculovirus, human adenovirus, SV40 or bovinepapilloma virus.
 25. The vector according to claims 23 or 24 furthercomprising a nucleotide sequence encoding at least one HLA class Imolecule.
 26. A host cell comprising the vector according to claim 23.27. The host cell according to claim 26 wherein the host celladditionally expresses an HLA class I molecule.
 28. The host cellaccording to claim 26 wherein the host cell is an antigen presentingcell.
 29. The host cell according to claim 28 wherein the host cell is adendritic cell.
 30. A method for treating a host having a tumorexpressing CEA or epitope thereof comprising introducing cytotoxic Tlymphocytes specific for CEA or epitope thereof to the host and at aperiodic interval thereafter introducing to the host an agonist peptideaccording to claim
 1. 31. The method according to claim 30 wherein thepeptide is selected from the group consisting of Seq ID Nos: 2, 3, 4,. 5or combination thereof.
 32. A method of inhibiting a CEAepitope-expressing carcinoma cells in a patient comprising administeringto said patient an effective amount of the peptide according to claim 1.33. The method according to claim 32 further comprising administrationof an immunostimulatory molecule.
 34. The method according to claim 33wherein the immunostimulatory molecule is selected from the groupconsisting of IL-2, B7.1, B7.2, ICAM-1, LFA-3, CD72, GM-CSF, TNFα, INFγ,IL-12, IL-6 and combinations thereof.
 35. The method according to claim32 further comprising administration of an adjuvant.
 36. The methodaccording to claim 32 wherein the carcinoma cell is gastrointestinal,breast, pancreatic, bladder, ovarian, lung, or prostate carcinoma cells.37. The method according to claim 32 further comprising theadministration of a vector comprising the gene encoding CEA.
 38. Amethod of inhibiting or killing CEA epitope-expressing carcinoma cellscomprising: A) generating CEA epitope or agonist peptide-specificcytotoxic T lymphocytes in vitro by stimulation of lymphocytes from asource with an effective amount of an agonist peptide according to claim1 alone or in combination with an immunostimulatory molecule; and B)adoptively transferring the CEA epitope or agonist peptide-specificcytotoxic T lymphocytes alone or in combination with the agonist peptideinto a mammal in an amount sufficient to inhibit or kill the CEA epitopeexpressing carcinoma cells.
 39. A method of inhibiting or killing CEAepitope-expressing carcinoma cells in a mammal comprising: A) generatingCEA epitope or agonist peptide-specific cytotoxic T lymphocytes in vivoby administration of an effective amount of a agonist peptide accordingto claim 1, an effective amount of a vector comprising a nucleic acidsequence encoding CEA or agonist peptide pulsed antigen presentingcells; and B) at a periodic interval providing the agonist peptideaccording to claim 1 alone or in combination with an adjuvant; whereinthe CEA epitope or agonist peptide-specific cytotoxic T lymphocytes sogenerated inhibit or kill CEA epitope-expressing carcinoma cells.
 40. Apeptide comprising an antagonist of a native sequence: YLSGANLNL (Seq.ID No: 1) wherein the antagonist varies at at least one amino acidposition from SEQ. ID No: 1 and the antagonist inhibits CEA-specificimmune responses.
 41. A pharmaceutical composition comprising thepeptide according to claim 36 and a pharmaceutically acceptable carrier.42. A method of inhibiting CEA-specific immune responses comprisingadministration of the peptide according to claim 40 in an amounteffective to inhibit the CEA-specific immune responses.
 43. The methodaccording to claim 42 wherein cytotoxic T lymphocytes specific for CEAor epitopes thereof are inhibited.